TY - JOUR
T1 - Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase
AU - Nakaar, Valerian
AU - Beckers, Con J.M.
AU - Polotsky, Vsevolod
AU - Joiner, Keith A.
N1 - Funding Information:
We thank members of the Joiner lab for helpful discussions on the manuscript. This work was supported by Public Health Service Grants AI-UO1-31808 and RO1-AI-30060 to K.A.J., who is also a Burroughs Wellcome Fund Scholar in Molecular Parasitology and V.N, a recipient of a NIH-Research Supplement for Underrepresented Minorities.
PY - 1998/11/30
Y1 - 1998/11/30
N2 - The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene family to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner KA. J Biol Chem 1994;269:29252-29260. Previous work also identified two isoforms of the enzyme in the virulent RH strain, and demonstrated that internal fragments of the genes encoding these isoforms were found differentially in virulent versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayashi H, Tsutomu T, J Biol Chem 1995;270:11391-11397). We now show that the NTPase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain lacks the gene for NTPase 3, explaining the absence of expression. Despite the fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid level, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cleaves predominantly nucleotide triphosphates. Furthermore, native and recombinant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agarose, further distinguishing the two isoforms. Using chimeras between the NTP1 and NTP3 genes, we show that a block of twelve residues at the C-terminus dictates substrate specificity. These residues lie outside the regions conserved among other E-ATPases, and therefore provide new insight into substrate recognition by this class of enzymes. Copyright (C) 1998 Elsevier Science B.V.
AB - The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene family to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner KA. J Biol Chem 1994;269:29252-29260. Previous work also identified two isoforms of the enzyme in the virulent RH strain, and demonstrated that internal fragments of the genes encoding these isoforms were found differentially in virulent versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayashi H, Tsutomu T, J Biol Chem 1995;270:11391-11397). We now show that the NTPase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain lacks the gene for NTPase 3, explaining the absence of expression. Despite the fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid level, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cleaves predominantly nucleotide triphosphates. Furthermore, native and recombinant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agarose, further distinguishing the two isoforms. Using chimeras between the NTP1 and NTP3 genes, we show that a block of twelve residues at the C-terminus dictates substrate specificity. These residues lie outside the regions conserved among other E-ATPases, and therefore provide new insight into substrate recognition by this class of enzymes. Copyright (C) 1998 Elsevier Science B.V.
KW - E-type ATPase
KW - Expression
KW - Nucleoside triphosphate hydrolase
KW - Substrate specificity
KW - Toxoplasma
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U2 - 10.1016/S0166-6851(98)00153-4
DO - 10.1016/S0166-6851(98)00153-4
M3 - Article
C2 - 9879899
AN - SCOPUS:0032583188
SN - 0166-6851
VL - 97
SP - 209
EP - 220
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1-2
ER -